Point-of-care testing for COVID-19: a simple two-step molecular diagnostic development and validation during the SARS-CoV-2 pandemic.

BACKGROUND: During the coronavirus disease 19 (COVID-19) pandemic, diagnostic testing of the general population proved challenging due to limitations of the gold-standard diagnostic procedure using reverse transcription real-time polymerase chain reaction (RT-qPCR) for large-scale testing on the centralised model, especially in low-resource areas. OBJECTIVES: To address this, a point-of-care (PoC) diagnostic protocol for COVID-19 was developed, providing fast, reliable, and affordable testing, particularly for low-mid develop areas. METHODS: The PoC diagnostic process combines a simple paper-based RNA extraction method housed within a 3D-printed plastic device with a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay. Nasopharyngeal/oropharyngeal swabs (NOS) and saliva samples were tested between 2020 and 2021, with the assistance of Santa Catarina's State Health Secretary, Brazil. FINDINGS: The developed diagnostic protocol showed a limit of detection of 9,900 copies and an overall diagnostic specificity of 98% for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from 1,348 clinical analysed samples. The diagnostic sensitivity was 95% for NOS samples, 85% for early morning saliva, and 69% for indiscriminate saliva. MAIN CONCLUSIONS: In conclusion, the developed device successfully extracted SARS-CoV-2 viral RNA from swabs and saliva clinical samples. When combined with colorimetric RT-LAMP, it provides results within 45 min using minimal resources, thus delivering a diagnostic kit protocol that is applicable in large-scale sampling.

Development and validation of RT-LAMP for detecting yellow fever virus in non-human primates samples from Brazil.

Monitoring yellow fever in non-human primates (NHPs) is an early warning system for sylvatic yellow fever outbreaks, aiding in preventing human cases. However, current diagnostic tests for this disease, primarily relying on RT-qPCR, are complex and costly. Therefore, there is a critical need for simpler and more cost-effective methods to detect yellow fever virus (YFV) infection in NHPs, enabling early identification of viral circulation. In this study, an RT-LAMP assay for detecting YFV in NHP samples was developed and validated. Two sets of RT-LAMP primers targeting the YFV NS5 and E genes were designed and tested together with a third primer set to the NS1 locus using NHP tissue samples from Southern Brazil. The results were visualized by colorimetry and compared to the RT-qPCR test. Standardization and validation of the RT-LAMP assay demonstrated 100% sensitivity and specificity compared to RT-qPCR, with a detection limit of 12 PFU/mL. Additionally, the cross-reactivity test with other flaviviruses confirmed a specificity of 100%. Our newly developed RT-LAMP diagnostic test for YFV in NHP samples will significantly contribute to yellow fever monitoring efforts, providing a simpler and more accessible method for viral early detection. This advancement holds promise for enhancing surveillance and ultimately preventing the spread of yellow fever.

Real-time qPCR coupled with high-resolution melting curve analysis for the detection of the internal transcribed spacer 1 of Angiostrongylus costaricensis.

Abdominal angiostrongyliasis (AA) is a zoonotic and severe parasitic infection caused by Angiostrongylus costaricensis. AA is currently diagnosed by the observation of A. costaricensis-compatible structures in biopsies or the detection of antibodies in serological tests. However, molecular methods targeting homologous sequences of A. costaricensis have not been designed before, and therefore, an HRM-coupled qPCR was developed to detect the internal transcribed spacer 1 (ITS1) of the parasite. The present assay successfully amplified DNA of A. costaricensis obtained from different hosts and identified slight sequence differences through the HRM analysis. The detection limit of the HRM-qPCR was 0.00036 ng/µL, 1.0 ng/µL, and 0.1 ng/µL when A. costaricensis DNA was diluted in nuclease-free water, whole blood, and sera, respectively, which highlights its potential use for cell-free DNA detection. Moreover, the reaction did not cross-amplify DNA of Angiostrongylus cantonensis, Strongyloides stercoralis, and other nematodes, thus emphasizing its specificity. Additionally, the assay tested positive in formalin-fixed paraffin embedded biopsies with visible A. costaricensis adults or eggs, but not in samples without evident parasites or a low number of larvae, which suggests that the reaction is useful for confirming the presence of the nematode in clinical samples. Finally, DNA of sera from patients with AA was evaluated with the HRM-qPCR but none tested positive, possibly due to long storage periods of the samples which could have led to cfDNA degradation. These results indicate that this assay may be useful in the confirmation of AA and its prospection for cell-free DNA detection protocols.

Loop-Mediated Isothermal Amplification for the Diagnosis of Sporotrichosis by Sporothrix brasiliensis.

We aimed to develop and validate a Loop-mediated Isothermal Amplification (LAMP) assay to Sporothrix brasiliensis. LAMP reaction was developed using six primers designed based on calmodulin gene. In the LAMP reaction, we tested twenty isolates of S. brasiliensis from animals and humans, along with ten tissue samples extracted from the left footpad of mice that had been experimentally infected with S. brasiliensis. In addition, it included DNA samples from various other fungal species for specificity evaluation. All S. brasiliensis isolates yielded positive results in the LAMP, and the limit of DNA detection was 1 ng/µL. All murine samples were positive in the test while DNA from other fungal species were all negative, resulting in 100% of sensitivity and specificity of primers. LAMP diagnosis technique is a promising alternative to sporotrichosis diagnosis, in a simple and cost-effective way. Further studies are warranted to validate this technique using animal model samples obtained from both humans and animals.

Development and accuracy evaluation of a new loop-mediated isothermal amplification assay targeting the HSP70 gene for the diagnosis of cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) is a global public health problem caused by species on the genus Leishmania and is the most prevalent clinical form of leishmaniasis. The aim of this study was to develop a new LAMP assay for Leishmania sp. based on HSP70 gene and evaluate it clinically for molecular diagnosis of CL. The study was carried out in the following stages: i) design of primers based on HSP70 gene of Leishmania sp.; ii) evaluation of detection limit and analytical specificity; iii) estimation of the accuracy of LAMP-Leish/HSP70 assay for diagnosing CL. A total of 100 skin biopsy samples from patients, comprising 60 CL cases and 40 non-cases, were analyzed in this study. One LAMP assay using HSP70 gene as molecular target were standardized, and the observed detection limit was 100fg of L. braziliensis purified DNA. The LAMP-Leish/HSP70 assay was specific for Leishmania spp. The LAMP-Leish/HSP70 assay showed an accuracy of 92%, and positivity rates were not affected by lesion onset time or parasite load. This novel LAMP assay targeting the HSP70 gene of Leishmania sp. has the potential to be a useful tool to integrate into routine diagnosis for suspected cases of CL.

Development and comparative assessment of RT-qPCR and duplex RT-LAMP assays for the monitoring of Aichi virus A (AiV-A) in untreated wastewater samples.

Diverse enteric pathogens, transmitted through human and animal feces, can cause gastroenteritis. Enteric viruses, such as human Aichi virus, specifically genotype A (AiV-A), are emerging pathogens that cause illnesses even at low doses and are spreading globally. This research developed a reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay targeting the 3CD junction and a reverse transcription colorimetric loop-mediated isothermal amplification (RT-cLAMP) duplex assay targeting junctions 2BC and 3CD of the AiV-A genome for rapid and sensitive detection of this virus in metropolitan and regional wastewater samples in Queensland, Australia. The performance of these assays was evaluated using control materials and by analyzing wastewater samples. In serially diluted control materials, RT-qPCR provided quantifiable data (mean 1.51 log10 GC/2 µL of nucleic acid) down to a dilution of 1 × 10-5 pg/µL. In comparison, the duplex RT-cLAMP assay detected down to 1 × 10-4 pg/µL, indicating that its sensitivity was one order of magnitude less than that of RT-qPCR. Of the 38 wastewater samples from 38 metropolitan and regional wastewater treatment plants (WWTPs) in Queensland, Australia, 21 (55.3 %) tested positive by RT-qPCR with concentrations ranging from 3.60 to 6.23 log10 GC/L. In contrast, only 15 (39.5 %) of 38 wastewater samples were positive using the duplex RT-cLAMP assay. The methods demonstrated substantial qualitative agreement (κ = 0.730), with a concordance of 86.5 %, demonstrating the reliability of RT-cLAMP for detecting AiV-A in wastewater samples. The duplex RT-cLAMP assay, despite demonstrating reduced detection sensitivity, has proven effective and holds promise as a supplementary approach, especially in settings with limited resources where rapid and affordable testing is crucial.

[Molecular diagnosis of bacteriaemia: benefits of using the FilmArray® BCID2 sepsis panel in a third level hospital]. / Diagnóstico molecular de bacteriemias: beneficios del empleo del panel de sepsis BCID2 de Filmarray® en un hospital de tercer nivel.

INTRODUCTION: Delay in initiating appropriate antimicrobial therapy prolongs hospitalization, increases in-hospital mortality, and raises economic costs. Currently, the identification and susceptibility testing of bacteria in positive blood cultures require a considerable amount of time. The objective of this study was to assess the impact of the BCID2 FilmArray® (FA) panel on the timing of appropriate antimicrobial therapy and potential antimicrobial costs. METHODS: This is a retrospective observational study focused on positive blood cultures in hospitalized patients. FA processing was conducted concurrently with routine sample processing. Changes in antibiotic treatments based on FA results were evaluated, and the reduction in antimicrobial therapy duration and associated cost savings were calculated. RESULTS: Eighty-seven bacteremia episodes were analysed. In 42 (48%) of them antimicrobial therapy was de-escalated to narrower spectrum agents, while in 7 (8%) therapy was escalated to broader spectrum antimicrobials. Additionally, in 8 (9%) antimicrobials were switched without changing spectrum and in 30 (34%) no changes were made based on FA results. Antimicrobial changes were made 2.3 days faster than with routine sample processing resulting in calculated potential savings of US$ 7408. CONCLUSION: The implementation of FA facilitated a faster administration of appropriate antimicrobial therapy, leading to a reduction in the duration of broadspectrum empirical antimicrobial therapy and subsequent economic savings.

Introducción: Los retrasos en el tratamiento antimicrobiano adecuado de las bacteriemias prolongan la estadía hospitalaria, aumentan la mortalidad e incrementan los costos. Aún hoy en día se requiere un tiempo considerable para obtener la identificación y antibiograma de los microorganismos en los hemocultivos positivos. El objetivo fue evaluar el impacto de la implementación del panel BCID2 de FilmArray® (FA) sobre el tiempo de inicio de tratamientos antimicrobianos adecuados y sobre los costos potenciales de los mismos. Métodos: Estudio observacional retrospectivo de los hemocultivos positivos de pacientes hospitalizados, procesados por FA y por metodología tradicional. Se evaluaron los cambios de antimicrobianos en base a los resultados del FA. Se calcularon los días de reducción de tratamiento antimicrobiano y el ahorro potencial en el uso de los mismos, teniendo en cuenta también los costos del FA. Resultados: Se analizaron 87 episodios de bacteriemia. En 42 (48.3%) de ellos se desescaló el tratamiento a antimicrobianos de menor espectro, en 7 (8%) se escaló a antimicrobianos de mayor espectro, en 8 (9.2%) se cambió el antimicrobiano sin variar el espectro y en 30 (34.5%) no se realizaron cambios con los resultados del FA. Los cambios de antimicrobianos se realizaron en promedio 2.3 días más rápido que con los métodos convencionales. Se calculó un ahorro potencial de US$ 7408. Conclusión: La implementación del panel BCID2 de FilmArray® permitió adecuar los tratamientos antimicrobianos más rápidamente acortando la duración de los tratamientos empíricos de amplio espectro, lo cual resultó costo-efectivo.

Aptamer-associated colorimetric reverse transcription loop-mediated isothermal amplification assay for detection of dengue virus.

Current diagnostic methods for dengue, such as serological tests, have limitations in terms of cross-reactivity with other viruses. To address this issue, we explored the potential of combining the loop-mediated isothermal amplification (LAMP) technique with the affinity of aptamers to develop point-of-care testing. In this study, we utilized 60 serum samples. An aptamer capable of binding to the dengue virus was employed as a platform for capturing genetic material, and its performance was compared to a commercial kit. Dengue virus was detected through RT-PCR and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP), allowing visual observation of the results without the need for equipment. In the context of the aptamer LAMP assay, our analysis revealed the detection of the dengue virus in 38 out of 60 samples, with 95% sensitivity and 100% specificity compared to RT-PCR and/or APTA-RT-PCR. Importantly, we observed no cross-reaction when assessing samples positive for the zika virus, underscoring the assay's selectivity. This innovative aptameric capture of the viral RNA in combination with the RT-LAMP (APTA-RT-LAMP) method has the potential to offer valuable molecular insights into neglected infectious diseases in a simpler and faster manner. IMPORTANCE: Dengue is a neglected tropical disease of significant epidemiological importance in tropical and subtropical countries. Current diagnostics for this infection present challenges, such as cross-reactivity in serological tests. Finding ways to enhance the diagnosis of this disease is crucial, given the absence of specific treatments. An accurate, simple, and effective diagnosis contributes to the improved management of infected individuals. In this context, our work combines molecular biology techniques, such as isothermal loop amplification, with aptamers to detect the dengue virus in biological samples. Our method produces colorimetric results based on a color change, with outcomes available in less than 2 hours. Moreover, it requires simpler equipment compared to molecular PCR tests.

Evaluation and validation of a PrintrLab-based LAMP assay to identify Trypanosoma cruzi in newborns in Bolivia: a proof-of-concept study.

BACKGROUND: Vertical transmission of Trypanosoma cruzi represents approximately 20% of new Chagas disease cases. Early detection and treatment for women of childbearing age and newborns is a public health priority, but the lack of a simple and reliable diagnostic test remains a major barrier. We aimed to evaluate the performance of a point-of-care loop-mediated isothermal amplification (LAMP) assay for the detection of T cruzi. METHODS: In this proof-of-concept study, we coupled a low-cost 3D printer repurposed for sample preparation and amplification (PrintrLab) to the Eiken T cruzi-LAMP prototype to detect vertically transmitted T cruzi, which we compared with standardised PCR and with the gold-standard algorithm (microscopy at birth and 2 months and serological study several months later). We screened pregnant women from two hospitals in the Bolivian Gran Chaco province, and those who were seropositive for T cruzi were offered the opportunity for their newborns to be enrolled in the study. Newborns were tested by microscopy, LAMP, and PCR at birth and 2 months, and by serology at 8 months. FINDINGS: Between April 23 and Nov 17, 2018, 986 mothers were screened, among whom 276 were seropositive for T cruzi (28·0% prevalence, 95% CI 25·6-31·2). In total, 224 infants born to 221 seropositive mothers completed 8 months of follow-up. Congenital transmission was detected in nine of the 224 newborns (4·0% prevalence, 1·9-7·5) by direct microscopy observation, and 14 more cases were diagnosed serologically (6·3%, 3·6-10·3), accounting for an overall vertical transmission rate of 10·3% (6·6-15·0; 23 of 224). All microscopy-positive newborns were positive by PrintrLab-LAMP and by PCR, while these techniques respectively detected four and five extra positive cases among the remaining 215 microscopy-negative newborns. INTERPRETATION: The PrintrLab-LAMP yielded a higher sensitivity than microscopy-based analysis. Considering the simpler use and expected lower cost of LAMP compared with PCR, our findings encourage its evaluation in a larger study over a wider geographical area. FUNDING: Inter-American Development Bank.

[A national network of molecular tests to detect Chlamydia trachomatis and Neisseria gonorrhoeae: a pilot implementation experience in Brazil]. / Rede nacional de testes moleculares para detecção de Chlamydia trachomatis e Neisseria gonorrhoeae: experiência de implantação-piloto no Brasil.

This study aimed to know the opinion of professionals participating in an experiment to implement a pilot for molecular tests to detect Chlamydia trachomatis and Neisseria gonorrhoeae at the Brazilian Unified National Health System (SUS). The detection rate of C. trachomatis and/or N. gonorrhoeae and the factors associated with infection were determined. The strategy included laboratories belonging to the HIV and viral hepatitis viral load network. Testing targeted people who are more vulnerable to sexually transmitted infections and collected urine samples and/or vaginal, endocervical, and/or male urethral swabs. Questionnaires were sent to state managers and laboratory professionals about the implementation of the pilot. Reviews were overall positive. Weaknesses included difficulties changing work processes, lack of human resources, poorly sensitized care professionals, and absence of primary urine tubes, the only input not provided. Strengths included the centralized acquisition of tests, sharing of equipment, and storage of samples at room temperature. Of the 16,177 people who were tested, 1,004 (6.21%) were positive for C. trachomatis; 1,036 (6.4%), for N. gonorrhoeae; and 239 (1.48%), for C. trachomatis/N. gonorrhoeae . Detection of any infection occurred more frequently in young people (≤ 24 vs. > 24 years) (adjOR = 2.65; 95%CI: 2.38-2.96), men (adjOR = 1.95; 95%CI: 1.72-2.21), brown/black individuals (adjOR = 1.06; 95%CI: 1.05-1.11), those in Southeastern Brazil (adjOR = 1.08; 95%CI: 1.02-1.13), and in urethral secretion samples (adjOR = 1.46; 95%CI: 1.41-1.52). Results show the importance of making testing available nationwide, which supported the implementation of a definitive network to detection C. trachomatis/N. gonorrhoeae in SUS.

O objetivo deste estudo foi conhecer a opinião dos profissionais participantes da implantação-piloto de testes moleculares para detecção de Chlamydia trachomatis e Neisseria gonorrhoeae no Sistema Único de Saúde (SUS). Determinou-se a taxa de detecção de C. trachomatis e/ou N. gonorrhoeae e os fatores associados à infecção. A estratégia contou com laboratórios pertencentes à rede de carga viral de HIV e hepatites virais. A testagem teve como público-alvo pessoas mais vulnerabilizadas às infecções sexualmente transmissíveis, com coleta de amostras de urina e/ou swabs vaginal, endocervical e/ou uretral masculino. Questionários foram enviados aos gestores estaduais e profissionais de laboratório sobre a implantação-piloto. De maneira geral, as avaliações foram positivas. Entre as fraquezas, citou-se dificuldades na mudança do processo de trabalho, carência de recursos humanos, pouca sensibilidade de profissionais da assistência e ausência de tubo primário de urina, único insumo não fornecido. Como fortaleza, destaca-se aquisição centralizada de testes, compartilhamento de equipamentos e armazenamento de amostras à temperatura ambiente. Das 16.177 pessoas testadas, 1.004 (6,21%) foram positivas para C. trachomatis, 1.036 (6,4%) para N. gonorrhoeae e 239 (1,48%) para C. trachomatis/N. gonorrhoeae. A detecção de infecção ocorreu mais em pessoas jovens (≤ 24 vs. > 24 anos) (aOR = 2,65; IC95%: 2,38-2,96), do sexo masculino (aOR = 1,95; IC95%: 1,72-2,21), pardas/pretas (aOR = 1,06; IC95%: 1,05-1,11), na Região Sudeste (aOR = 1,08; IC95%: 1,02-1,13) e em amostras de secreção uretral (aOR = 1,46; IC95%: 1,41-1,52). Os resultados deste estudo demonstraram a importância da disponibilização da testagem em âmbito nacional, os quais subsidiaram a implantação da rede definitiva para detecção de C. trachomatis/N. gonorrhoeae no SUS.

El objetivo de este estudio fue conocer la opinión de los profesionales participantes de la implantación piloto de pruebas moleculares para la detección de Chlamydia trachomatis y Neisseria gonorrhoeae en el Sistema Único de Salud brasileño (SUS). Se determinó la tasa de detección de C. trachomatis y/o N. gonorrhoeae y los factores asociados con la infección. En la estrategia participaron laboratorios pertenecientes a la red de carga viral de VIH y hepatitis virales. La prueba tuvo como público objetivo a personas más vulnerables a las infecciones de transmisión sexual, con recolección de muestras de orina y/o swabs vaginal, endocervicales y/o uretral masculino. Se enviaron cuestionarios a los gestores estatales y a los profesionales de laboratorio sobre la implementación piloto. En general, las evaluaciones fueron positivas. Entre las debilidades, se citó las dificultades en el cambio del proceso de trabajo, la falta de recursos humanos, los profesionales de la asistencia poco sensibilizados y la ausencia del contenedor de orina primaria, el único insumo no suministrado. Como fortalezas, se destaca la adquisición centralizada de pruebas, el intercambio de equipos y el almacenamiento de muestras a temperatura ambiente. De las 16.177 personas evaluadas, 1.004 (6,21%) fueron positivas para C. trachomatis, 1.036 (6,4%) para N. gonorrhoeae y 239 (1,48%) para C. trachomatis/N. gonorrhoeae. La detección de alguna infección ocurrió más en personas jóvenes (≤ 24 vs. > 24 años) (aOR = 2,65; IC95%: 2,38-2,96), del sexo masculino (aOR = 1,95; IC95%: 1,72-2,21), parda/negra (aOR = 1,06; IC95%: 1,05-1,11), localizadas en la región Sudeste (aOR = 1,08; IC95%: 1,02-1,13) y en muestras de secreción uretral (aOR = 1,46; IC95%: 1,41-1,52). Los resultados de este estudio demostraron la importancia de la disponibilidad de la prueba a nivel nacional, los cuales subsidiaron la implantación de la red definitiva para detección de C. trachomatis/N. gonorrhoeae en el SUS.

Sporothrix brasiliensis-specific polymerase chain reaction for the diagnosis of cat and human sporotrichosis through non-invasive samples.

Zoonotic sporotrichosis caused by Sporothrix brasiliensis is an emerging mycosis in Latin America. One of the problems to quickly treat infected animals and break the transmission chain is associated with the time-consuming gold-standard diagnosis method (culture). We aimed to evaluate a species-specific polymerase chain reaction (PCR) for the diagnosis of sporotrichosis caused by S. brasiliensis using non-invasive samples. We performed a retrospective cross-sectional study using samples collected with swabs from humans and cats with clinical suspicion of sporotrichosis. Deoxyribonucleic acid (DNA) was extracted using a commercial kit, and a species-specific PCR for S. brasiliensis detection was performed. One hundred ten samples were included. PCR showed a good concordance with culture (86% of agreement) for human and cat samples (Kappa coefficient = 0.722, and 0.727, respectively). In conclusion, our data shows that this adapted PCR using non-invasive samples can be applied to sporotrichosis diagnosis, being a good alternative mainly in regions with a lack of mycologists to identify the fungus in culture, contributing to the control of this emergent zoonosis.

We aimed to evaluate a molecular method for diagnosing sporotrichosis caused by Sporothrix brasiliensis in humans and cats. We observed that the technique is in good agreement with the classic method and is a good alternative for assisting in the diagnosis and consequent control of this zoonosis.

Optimization of the molecular detection assay of Salmonella (MDS®) for drag swab samples of aviaries.

Detection of Salmonella sp. is important for the broiler chicken production chain because it is one microorganisms involved in food-borne diseases. Thus, this study performed the optimization of a technique of Loop-mediated isothermal DNA amplification (LAMP) through the 3MTM Molecular Detection Assay 2: Salmonella (MDS®), in accordance with Ordinance number 126 of the Ministry of Agriculture, for the detection of Salmonella sp. in drag swab. The methodology followed ISO 16140-2: 2016, with the analysis naturally contaminated drag swab samples collected from broiler aviaries and artificially contaminated with salmonella ATCCs. Of the 300 samples processed in protocol A (pre-enrichment tetrathionate broth (TT)), 45 were positive for Salmonella sp., 242 negative, one false-positive, and 12 false-negative, while of the 300 samples analyzed in protocol B (pre-enrichment brain-heart infusion broth (BHI)), 40 were positive, 256 negative, one false-positive, and three false-negative. The result for protocol A was a sensitivity of 79%, specificity of 99.6%, Positive Predictive Value (PPV) of 98%, and Negative Predictive Value (NPV) of 95%; and for protocol B, 93% sensitivity, 99.6% specificity, 98% PPV, and 99% NPV. Both protocols were associated with the reference method (p>0.05), concluding that the MDS® can be used for the qualitative detection of Salmonella sp.

Evaluation of self-collected nasal, urine, and saliva samples for molecular detection of SARS-CoV-2 using an EUA approved RT-PCR assay and a laboratory developed LAMP SARS-CoV-2 test.

As the SARS-CoV-2 virus spread throughout the world, millions of positive cases of COVID-19 were registered and, even though there are millions of people already vaccinated against SARS-CoV-2, a large part of the global population remains vulnerable to contracting the virus. Massive nasopharyngeal sample collection in Puerto Rico at the beginning of the pandemic was limited by the scarcity of trained personnel and testing sites. To increase SARS-CoV-2 molecular testing availability, we evaluated the diagnostic accuracy of self-collected nasal, saliva, and urine samples using the TaqPath reverse transcription polymerase chain reaction (RT-PCR) COVID-19 kit to detect SARS-CoV-2. We also created a colorimetric loop-mediated isothermal amplification (LAMP) laboratory developed test (LDT) to detect SARS-CoV-2, as another strategy to increase the availability of molecular testing in community-based laboratories. Automated RNA extraction was performed in the KingFisher Flex instrument, followed by PCR quantification of SARS-CoV-2 on the 7500 Fast Dx RT-PCR using the TaqPath RT-PCR COVID-19 molecular test. Data was interpreted by the COVID-19 Interpretive Software from Applied Biosystems and statistically analyzed with Cohen's kappa coefficient (k). Cohen's kappa coefficient (k) for paired nasal and saliva samples showed moderate agreement (0.52). Saliva samples exhibited a higher viral load. We also observed 90% concordance between LifeGene-Biomarks' SARS-CoV-2 Rapid Colorimetric LAMP LDT and the TaqPath RT-PCR COVID-19 test. Our results suggest that self-collected saliva is superior to nasal and urine samples for COVID-19 testing. The results also suggest that the colorimetric LAMP LDT is a rapid alternative to RT-PCR tests for the detection of SARS-CoV-2. This test can be easily implemented in clinics, hospitals, the workplace, and at home; optimizing the surveillance and collection process, which helps mitigate global public health and socioeconomic upheaval caused by airborne pandemics.

Operational indicators for pulmonary tuberculosis diagnosis in people living with HIV before and after Xpert MTB/RIF implementation in the state of São Paulo, Brazil.

Tuberculosis (TB) in people living with HIV (PLHIV) is usually paucibacillary and the smear microscopy has limitations and may lead to high proportions of non-confirmed pulmonary tuberculosis (NC-PTB). Despite culture being the reference method, it usually takes 6 to 8 weeks to produce the results. This study aimed to analyze the effect of a rapid molecular test (Xpert) in the confirmatory rate of PTB among PLHIV, from 2010 to 2020, in São Paulo state, Brazil. This is an ecological study with time series analysis of the trend and the NC-PTB rates before and after Xpert implementation in 21 municipalities. The use of Xpert started and gradually increased after 2014, while the rate of NC-PTB in PLHIV decreased over this time, being more significant between late 2015 and mid-2017. The city of Ribeirão Preto stands out for having the highest percentage (75.0%) of Xpert testing among PLHIV and for showing two reductions in the NC-PTB rate. The cities with low Xpert coverage had a slower and smaller decrease in the NC-PTB rate. Despite being available since 2014, a significant proportion of PLHIV suspected of PTB in the state of São Paulo did not have an Xpert ordered by the doctors. The implementation of Xpert reduced the NC-PTB rates with growing effect as the coverage increased in the municipality.

Findings and Challenges in Replacing Traditional Uterine Cervical Cancer Diagnosis with Molecular Tools in Private Gynecological Practice in Mexico.

We have been encouraging practicing gynecologists to adopt molecular diagnostics tests, PCR, and cancer biomarkers, as alternatives enabled by these platforms, to traditional Papanicolaou and colposcopy tests, respectively. An aliquot of liquid-based cytology was used for the molecular test [high-risk HPV types, (HR HPV)], another for the PAP test, and one more for p16/Ki67 dual-stain cytology. A total of 4499 laboratory samples were evaluated, and we found that 25.1% of low-grade samples and 47.9% of high-grade samples after PAP testing had a negative HR HPV-PCR result. In those cases, reported as Pap-negative, 22.1% had a positive HR HPV-PCR result. Dual staining with p16/Ki67 biomarkers in samples was positive for HR HPV, and 31.7% were also positive for these markers. Out of the PCR results that were positive for any of these HR HPV subtypes, n 68.3%, we did not find evidence for the presence of cancerous cells, highlighting the importance of performing dual staining with p16/Ki67 after PCR to avoid unnecessary colposcopies. The encountered challenges are a deep-rooted social reluctance in Mexico to abandon traditional Pap smears and the opinion of many specialists. Therefore, we still believe that colposcopy continues to be a preferred procedure over the dual-staining protocol.

A Highly Sensitive Molecular Technique for RNA Virus Detection.

Zika (ZIKV) and Chikungunya (CHIKV) viruses are mosquito-transmitted infections, or vector-borne pathogens, that emerged a few years ago. Reliable diagnostic tools for ZIKV and CHIKV-inexpensive, multiplexed, rapid, highly sensitive, and specific point-of-care (POC) systems-are vital for appropriate risk management and therapy. We recently studied a detection system with great success in Mexico (Villahermosa, state of Tabasco), working with human sera from patients infected with those viruses. The research conducted in Mexico validated the efficacy of a novel two-step rapid isothermal amplification technique (RAMP). This approach, which encompasses recombinase polymerase amplification (RPA) followed by loop-mediated isothermal amplification (LAMP), had been previously established in the lab using lab-derived Zika (ZIKV) and Chikungunya (CHIKV) viruses. Crucially, our findings confirmed that this technique is also effective when applied to human sera samples collected from locally infected individuals in Mexico.

Universal and Expanded Screening Strategy for Congenital Cytomegalovirus Infection: Is Pool Testing by a Rapid Molecular Test in Saliva a New Choice in Developing Countries?

BACKGROUND: Several screening strategies for identifying congenital CMV (cCMV) have been proposed; however, the optimal solution has yet to be determined. We aimed to determine the prevalence of cCMV by universal screening with saliva pool testing and to identify the clinical variables associated with a higher risk of cCMV to optimize an expanded screening strategy. METHODS: We carried out a prospective universal cCMV screening (September/2022 to August/2023) of 2186 newborns, analyzing saliva samples in pools of five (Alethia-LAMP-CMV®) and then performed confirmatory urine CMV RT-PCR. Infants with risk factors (small for gestational age, failed hearing screening, HIV-exposed, born to immunosuppressed mothers, or <1000 g birth weight) underwent expanded screening. Multivariate analyses were used to assess the association with maternal/neonatal variables. RESULTS: We identified 10 infants with cCMV (prevalence: 0.46%, 95% CI 0.22-0.84), with significantly higher rates (2.1%, 95% CI 0.58-5.3) in the high-risk group (p = 0.04). False positives occurred in 0.09% of cases. No significant differences in maternal/neonatal characteristics were observed, except for a higher prevalence among infants born to non-Chilean mothers (p = 0.034), notably those born to Haitian mothers (1.5%, 95% CI 0.31-4.34), who had higher odds of cCMV (OR 6.82, 95% CI 1.23-37.9, p = 0.04). Incorporating maternal nationality improved predictive accuracy (AUC: 0.65 to 0.83). CONCLUSIONS: For low-prevalence diseases such as cCMV, universal screening with pool testing in saliva represents an optimal and cost-effective approach to enhance diagnosis in asymptomatic patients. An expanded screening strategy considering maternal nationality could be beneficial in resource-limited settings.

Development and evaluation of a lyophilization protocol for colorimetric RT-LAMP diagnostic assay for COVID-19.

Molecular diagnostics involving nucleic acids (DNA and RNA) are regarded as extremely functional tools. During the 2020 global health crisis, efforts intensified to optimize the production and delivery of molecular diagnostic kits for detecting SARS-CoV-2. During this period, RT-LAMP emerged as a significant focus. However, the thermolability of the reagents used in this technique necessitates special low-temperature infrastructure for transport, storage, and conservation. These requirements limit distribution capacity and necessitate cost-increasing adaptations. Consequently, this report details the development of a lyophilization protocol for reagents in a colorimetric RT-LAMP diagnostic kit to detect SARS-CoV-2, facilitating room-temperature transport and storage. We conducted tests to identify the ideal excipients that maintain the molecular integrity of the reagents and ensure their stability during room-temperature storage and transport. The optimal condition identified involved adding 5% PEG 8000 and 75 mM trehalose to the RT-LAMP reaction, which enabled stability at room temperature for up to 28 days and yielded an analytical and diagnostic sensitivity and specificity of 83.33% and 90%, respectively, for detecting SARS-CoV-2. This study presents the results of a lyophilized colorimetric RT-LAMP COVID-19 detection assay with diagnostic sensitivity and specificity comparable to RT-qPCR, particularly in samples with high viral load.

Rapid and visual detection of Shiga-toxigenic Escherichia coli (STEC) in carabeef meat harnessing loop-mediated isothermal amplification (LAMP).

Shiga toxigenic E. coli are important foodborne zoonotic pathogens. The present study was envisaged to standardize loop-mediated isothermal amplification assays targeting stx1 and stx2 genes for rapid and visual detection of STEC and compare its sensitivity with PCR. The study also assessed the effect of short enrichment on the detection limit of LAMP and PCR. The developed LAMP assays were found to be highly specific. Analytical sensitivity of LAMP was 94 fg/µLand 25.8 fg/µL for stx-1 and stx-2 while LOD of 5 CFU/g of carabeef was measured after 6-12 h enrichment. The study highlights the importance of short (6-12 h) enrichment for improving the sensitivity of LAMP. The entire detection protocol could be performed within 9 h yielding results on the same day. The developed LAMP assays proved to be a handy and cost-effective alternative for screening STEC contamination in meat.

Sampling is decisive to determination of Leishmania (Viannia) species.

BACKGROUND: Accuracy of molecular tools for the identification of parasites that cause human cutaneous leishmaniasis (CL) could largely depend on the sampling method. Non-invasive or less-invasive sampling methods such as filter paper imprints and cotton swabs are preferred over punch biopsies and lancet scrapings for detection methods of Leishmania based on polymerase chain reaction (PCR) because they are painless, simple, and inexpensive, and of benefit to military and civilian patients to ensure timely treatment. However, different types of samples can generate false negatives and there is a clear need to demonstrate which sample is more proper for molecular assays. METHODOLOGY: Here, we compared the sensitivity of molecular identification of different Leishmania (Viannia) species from Peru, using three types of sampling: punch biopsy, filter paper imprint and lancet scraping. Different composite reference standards and latent class models allowed to evaluate the accuracy of the molecular tools. Additionally, a quantitative PCR assessed variations in the results and parasite load in each type of sample. PRINCIPAL FINDINGS: Different composite reference standards and latent class models determined higher sensitivity when lancet scrapings were used for sampling in the identification and determination of Leishmania (Viannia) species through PCR-based assays. This was consistent for genus identification through kinetoplastid DNA-PCR and for the determination of species using FRET probes-based Nested Real-Time PCR. Lack of species identification in some samples correlated with the low intensity of the PCR electrophoretic band, which reflects the low parasite load in samples. CONCLUSIONS: The type of clinical sample can directly influence the detection and identification of Leishmania (Viannia) species. Here, we demonstrated that lancet scraping samples consistently allowed the identification of more leishmaniasis cases compared to filter paper imprints or biopsies. This procedure is inexpensive, painless, and easy to implement at the point of care and avoids the need for anesthesia, surgery, and hospitalization and therefore could be used in resource limited settings for both military and civilian populations.